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UNLABELLED: The interaction between resident cells and electrospun nanofibers is critical in determining resultant osteoblast proliferation and activity in orthopedic tissue scaffolds. The use of techniques to evaluate cell-nanofiber interactions is critical in understanding scaffold function, with visualization promising unparalleled access to spatial information on such interactions. 3D tomography exploiting focused ion beam (FIB)-scanning electron microscopy (SEM) was used to examine electrospun nanofiber scaffolds to understand the features responsible for (osteoblast-like MC3T3-E1 and UMR106) cell behavior and resultant scaffold function. 3D imaging of cell-nanofiber interactions within a range of electrospun poly(d,l-lactide-co-glycolide acid) (PLGA) nanofiber scaffold architectures indicated a coherent interface between osteoblasts and nanofiber surfaces, promoting osteoblast filopodia formation for successful cell growth. Coherent cell-nanofiber interfaces were demonstrated throughout a randomly organized and aligned nanofiber network. Gene expression of UMR106 cells grown on PLGA fibers did not deviate significantly from those grown on plastic, suggesting maintenance of phenotype. However, considerably lower expression of Ibsp and Alpl on PLGA fibers might indicate that these cells are still in the proliferative phase compared with a more differentiated cell on plastic. This work demonstrates the synergy between designing electrospun tissue scaffolds and providing comprehensive evaluation through high resolution imaging of resultant 3-dimensional cell growth within the scaffold. STATEMENT OF SIGNIFICANCE: Membranes made from electrospun nanofibers are potentially excellent for promoting bone growth for next-generation tissue scaffolds. The effectiveness of an electrospun membrane is shown here using high resolution 3D imaging to visualize the interaction between cells and the nanofibers within the membrane. Nanofibers that are aligned in one direction control cell growth at the surface of the membrane whereas random nanofibers cause cell growth into the membrane. Such observations are important and indicate that lateral cell growth at the membrane surface using aligned nanofibers could be used for rapid tissue repair whereas slower but more extensive tissue production is promoted by membranes containing random nanofibers.